Measurements of oxygen consumption and generation of reactive oxygen species (ROS) in cardiomyocytes, assume importance in pathophysiology of ischemic heart disease because large quantities of oxygen free radicals are generated during ischemia/reperfusion, and the produced free radicals impair the heart function. Though the oxygen consumption and free radicals generation occur simultaneously, till date there is no method known to simultaneously measure both oxygen consumption and free radicals generation in a single experiment. The purpose of the present grant proposal is to explore the application of a new EPR technique called "Microxymetry" to achieve this goal, i.e., the real time quantitation of both the oxygen consumption and free radicals generation, in a single experiment. The present approach combines the EPR oxymetry and spin trapping into one, to get both oxygen concentration and free radicals concentration in one step. The specific aims of the proposal are: (i) to study the respiration (both as such and stimulated by added stimulants) of cardiomyocytes (both cultured cells and freshly isolated from perfused hearts) at different conditions. Particularly, in these systems, how the simulated ischemia alters oxygen consumption rate of cardiomyocytes will be studied in detail. The ROS generated during the respiration of these ceils at physiologically altered conditions, will be studied simultaneously and the relationship between oxygen consumption and the amount of ROS formed will be evaluated at various deleterious conditions like hypoxia. Further, the effects of simulated ischemic preconditioning and induction of heat shock proteins on the oxygen consumption rate and ROS production will also be studied using the proposed microxymetry; (ii) to study the respiration of isolated heart mitochondria using the proposed microxymtery. Similar to the cardiomyocytes, the heart mitochondria will be subjected to different conditions like hypoxia and normoxiaI PC cycles and the effect of these treatments on mitochondria respiration will be studied. It is also proposed to use this technique to study the specific inhibitors on complex I and complex Ill and their effect on the state 3 and state 4 respirations and the subsequent influences on the oxygen consumption of mitochondria. Overall, by establishing this new technique, it is possible to simultaneously measure the oxygen consumption and ROS production in microliter volumes of cellular and sub-cellular components.